The invention relates generally to the field of mass spectrometry. In particular, the invention relates to a method and apparatus for electrodynamic ion trap mass spectrometry.
Identification of molecular species by successive reactions in a mass spectrometer is known as xe2x80x9cmass spectrometry/mass spectrometry,xe2x80x9d xe2x80x9cmultidimensional mass spectrometry,xe2x80x9d or more commonly xe2x80x9cMS/MS,xe2x80x9d or xe2x80x9cMSn. xe2x80x9d In this process, an analyte ion usually decomposes spontaneously or is induced to fragment between stages of mass analysis. The process is executed by selecting an ion of specific mass-to-charge ratio (m/z) value and measuring the m/z value(s) of the fragment ions derived therefrom. Fragments of an ion are highly specific for the parent ion from which they are derived.
In a further exploitation of this process, a first generation fragment ion derived from a specific parent may be further fragmented and the second generation fragment ions mass analyzed. The number of ions available for analysis declines in each successive stage of fragmentation. The rate of decline depends upon the ion transmission characteristics of the mass spectrometer and the number and relative abundances of fragment ions in each stage. Sequential fragmentation reactions may be continued until the number of ions formed is below the detection level of the mass spectrometer being used. Fragmentation reactions constitute an important class of reactions in MS/MS. However, a variety of other types of reactions involving reactions of ions with molecules or with other ions can also be used between stages of mass analysis.
Electrospray ionization (ESI) is a process by which small droplets of liquid are sprayed from a charged capillary. These droplets are generally highly charged. As liquid evaporates from the sprayed droplets, they become smaller and the charge density increases. When the charge density is sufficiently high, droplets are further fragmented into smaller droplets by charge repulsion in the droplets. This cycle of evaporation and fragmentation by electrostatic repulsion continues until the charge density on the surface is sufficiently high that ions on the surface can desorb into the gas phase.
ESI is particularly effective in yielding multiply charged ions from species that can accommodate more than a single charge. Multiple charging is particularly common in proteins containing large numbers of free amine groups. For example, it would be possible in a protein containing 30 amine groups to exist as positive ions with a distribution of charge states in the range of +12 to +20, including species at every charge state within the range. The singly charged ion, however, is generally not observed because ions of such low charge state (z=1) are not typically formed via ESI. Because mass spectrometry separates ions on the basis of m/z, each of the charge states of the intact protein will produce a separate peak in a mass spectrometer. In the example of the protein described above, if the intact protein had a molecular weight of 20,000 Daltons (Da), ions would be measured at m/z=1000.00, m/z=1052.6, m/z=1111.1, m/z=1176.5, m/z=1250, m/z=1333.3, m/z=1428.6, m/z=1538.5, and m/z=1666.7. The molecular weight of the intact protein is obtained by using an algorithm that computes the probable molecular weight from the observed charge state distribution given by the peaks of the mass spectrum.
Since ESI produces multiple ions of varying charge states, analyzing mixtures of molecules is problematic, especially for mixtures of proteins. Even mixtures with a small number of species will produce so many ions that it is not possible to associate the various ions with the individual molecules from which they were derived. In addition, multiple charging compresses the xe2x80x9cmass scale,xe2x80x9d that is, the distance between adjacent charge states on the m/z scale decreases with increasing charge, and further increase the difficulty of resolving molecules in a mixture.
Further, it is not uncommon for some of the charge states of molecules of different mass to have m/z values that are too similar to be resolved by the mass spectrometer. For example, an ion with a mass of 10,000 Da in a z=20 charge state will have substantially the same m/z value as an 5,000 Da ion in a z=10 charge state. Thus, the multiple charging phenomenon gives rise to the possibility that two molecules of different mass can give rise to ions with similar m/z values, thereby further complicating the analysis of a mixture of the molecules. For this reason, extensive efforts are usually undertaken to introduce relatively pure molecules, and in particular pure proteins, one molecular species at a time to an ESI ion source. These efforts ordinarily involve time-consuming off-line and on-line separations, severely limiting sample throughput.
The problem of multiple charging associated with ESI of mixtures has been addressed through charge quenching reactions. There are two general approaches by which charge quenching reactions can be effected. One approach involves mixing ions of opposite polarity in a region with minimal external electric or magnetic fields. This approach is exemplified by mixing ions of opposite polarity external to a mass spectrometer and sampling the charge quenched ions into the mass spectrometer for mass analysis. This approach constitutes a straightforward single stage mass spectrometry experiment and is not amenable to MS/MS or MSn procedures. The other general approach allows ions of opposite polarity to interact within combined electrostatic and magnetic fields or within an electrodynamic field, such as provided by electrodynamic ion traps. The latter approach allows for greater overlap in space of the oppositely charged ions.
In either general charge quenching approach, after ionization but before mass analysis, the charges of all ionic species are quenched to a single charge in the gas phase. Subsequent to charge quenching, the mixture is mass analyzed. This process substantially reduces the number of charged species in the gas phase before analysis and greatly simplifies the mass spectrum. Peaks in the spectrum appear at an m/z values equivalent to the molecular weight of the protein plus the mass of a proton.
The charge quenching process significantly improves the mixture analysis capabilities of electrospray. However, in many protein mixture analysis strategies, it is desirable to detect and quantify molecular species present at a wide range of concentrations. The concentration range over which mixture components can be measured is often referred to as xe2x80x9cdynamic range.xe2x80x9d Thus, an accurate and reliable method of charge quenching over a large dynamic range is desirable.
What is needed is a mass spectrometry method and apparatus that improves the dynamic range, signal discrimination, and throughput of samples ionized by electrospray ionization.
The invention provides methods and apparatus that improve the sample throughput, dynamic mass range and signal discrimination in the mass spectrometry of multiply charged ions. The invention improves the dynamic range associated ESI of protein mixtures by as much as four orders of magnitude. The above advantages are of particular importance in the mass analysis of mixtures of molecules. In particular, the mass analysis of mixtures of biomolecules, including, but not limited to, proteins, peptides, carbohydrates, and oligonucleotides, can benefit from the invention.
The invention provides a method of mass spectrometry in which multiply charged ionic species are admitted into and/or retained in an electrodynamic ion trap in a mass to charge-ratio dependent (m/z-dependent) fashion and then partially charge quenched and subsequently mass analyzed. The procedure is repeated as a function of mass and allows for the measurement and quantification of multiple molecular species in a highly complex mixture. The methods of the invention provide for the detection of molecules of relatively low abundance in a mixture. For example, in one embodiment, ion trap accumulation times are varied to, for example, enhance the signals of low abundance molecular species ordinarily obscured by signals from much more abundant species.
In one aspect, the invention provides methods for analyzing a sample of molecules to obtain a mass spectrum of the sample. In particular, the methods of the invention are useful where an ion source produces a plurality of multiply charge ions from a sample. Such ion sources include, but are not limited to, electrospray ionization, laser desorption, and matrix assisted laser desorption ionization (MALDI) sources. The invention provides a charge quenching process that, in conjunction with a tailored waveform, serves to filter out ions of the same m/z value but with different mass. The methods of the invention can successively segregate subsets of ions from a mixture of multiply charged sample ions and then partially quench the charge state of the subset ions to produce a mass spectrum of the mixture with improved dynamic range and signal discrimination.
More specifically, a tailored waveform is used to segregate a subset of ions by applying the tailored waveform to an electrodynamic ion trap such that only ions in the subset are allowed into and/or are retained in the ion trap. That is, the tailored waveform allows only ions within select mass-to-charge ratio ranges into and/or to remain in the ion trap. In one embodiment, the subset of ions includes ions within mass-to-charge ratio (m/z) value ranges where the median values of the ranges are substantially the m/z values of the integer charge states of a select ion mass. For example, if the ion mass of interest is 10,000 Daltons (Da), the tailored waveform is created to allow and/or retain in the ion trap only m/z value ranges (xe2x80x9cm/z rangesxe2x80x9d) with median values that correspond to the charge states of a 10,000 Da ion, e.g., median values of approximately m/z=10,000, m/z=5,000, m/z=3333.33, m/z=2,500 and so forth. The width of the m/z ranges are chosen, for example, based on the m/z values of the ions of interest, how the molecules are charged, the range of the m/z scale of interest, ion abundance, experimental protocol, instrumentation limitations, or investigator convenience. Preferable, the m/z ranges are chosen such that they do not significantly overlap. The subset ions allowed into and/or retained in the ion trap are then reacted with a quencher to lower the charge state of the ions in the ion trap. The quencher can be a neutral or have a charge, and can be an atom or a molecule. Preferably, the quencher is an ion of opposite polarity to that of the subset ions. After the ions in the ion trap have been reacted with a quencher, the resulting ions are released from the ion trap and a mass signal is determined for the highest mass-to-charge ratio ion by any suitable mass spectrometer or series of mass spectrometers. Suitable mass spectrometers include, but are not limited to, time-of-flight, quadrupole, Wein filter, magnetic sector, and electrostatic sector instruments.
In one embodiment, the ion of highest m/z value corresponds to the lowest charge state of the ion mass of interest (z=1). However, it is to be understood that depending on the reaction time and reaction rate between the ions and the quencher, the ions released from the ion trap may include, in addition to the highest m/z value ion, other lower order m/z ions, e.g., the second lowest (z=2), and/or the third lowest (z=3). According to certain embodiments of methods of the invention, after obtaining a mass signal for one ion mass of interest, the tailored waveform is varied to obtain a mass signal for at least one other ion mass of interest. In this manner, the methods of the invention can obtain a mass spectrum of a sample with improved dynamic range and signal discrimination.
In other embodiments, the invention also provides methods which increase dynamic mass range, signal discrimination, and/or signal-to-noise ratios, in an efficient manner conducive to high throughput sample analysis. In one embodiment, a tailored waveform is used to segregate a subset of ions by applying the waveform to an electrodynamic ion trap such that only ions in the subset are allowed into and/or are retained in the ion trap for an accumulation time. The subset of ions allowed into and/or retained in the ion trap are then reacted with a quencher to partially lower the charge state of the ions in the ion trap. After the ions in the ion trap have been reacted with the quencher, the ions are released from the ion trap and a mass signal is determined for the highest m/z value ion by any suitable mass spectrometer or series of mass spectrometers.
In one embodiment, if the mass signal intensity is too weak, the process is repeated with substantially the same tailored waveform for a longer accumulation time to increase the signal intensity. In another embodiment, if the mass signal intensity is too high, the process is repeated with substantially the same tailored waveform for a shorter accumulation time to decrease the signal intensity. In another embodiment, the accumulation time varies with the ion mass of interest based on, for example, the importance of the ion mass, analysis protocol, and/or known or suspected ion mass source. For example, some ion masses may only be of interest if they have a signal level above a certain threshold, such as those associated with certain food contaminants. Other ion masses may be of particular interest and warrant longer accumulation times, such as those corresponding to early markers for disease. Still other ion masses may be of little interest and warrant minimal accumulation times, such as those corresponding to known contaminants or experimental artifacts. In this manner the invention can obtain a mass spectrum of a sample with improved dynamic mass range, signal discrimination, and/or signal-to-noise ratios, in an efficient manner conducive to high throughput sample analysis.
In other embodiments, the invention provides methods of charge quenching in conjunction with the use of a tailored waveform. These embodiments can improve the distinction between ions of the same m/z value but different mass, while also improving dynamic mass range and signal discrimination. In one embodiment, a primary tailored waveform is used to segregate a first subset of ions by applying the primary tailored waveform to an electrodynamic ion trap such that only ions in a first subset are allowed into and/or are retained in the ion trap. The first subset of ions allowed in to and/or retained in the ion trap are then reacted with a quencher to partially lower the charge state of the ions in the ion trap. A secondary tailored waveform is then used to retain in the ion trap, a second subset of ions, which includes a subset of the first subset of ions. For example, the second subset of ions may include, for the ion mass of interest, only a certain charge state(s) of the charge states initially selected by the primary tailored waveform. This charge state(s) need not be the lowest charge state of the ion mass of interest and may comprise any combination of charge states.
For example, a secondary tailored waveform may retain both the lowest charge state and higher charge states. That is, a secondary tailored waveform could be generated to retain the lowest charge state (z=1), yet still retain select higher charge states which may contain a significant population of the ion mass of interest. The select higher charge states can be chosen, for example, based on the non-linear dependence of the reaction rate between ions and an ionic quencher of opposite polarity. After the application of the secondary tailored waveform, the ions are then released from the ion trap and a mass signal is determined for the highest m/z value ion by any suitable mass spectrometer or series of mass spectrometers. After obtaining a mass signal for one ion mass of interest, both the primary and secondary tailored waveforms may be varied to obtain a mass signal for at least one other ion mass of interest.
In another embodiment, the second subset of ions are also reacted with a quencher, which can be the same quencher as reacted with the first subset of ions or a different quencher. For example, the quencher reacted with the first subset of ions could be an ionic quencher of opposite polarity to take advantage of the non-linear dependence of ionxe2x80x94ion reaction rates on ion charge, while the quencher reacted with the second subset of ions could be a neutral species to avoid the non-linear dependence of ionxe2x80x94ion reaction rates. In another example, the quencher reacted with the second subset of ions could have a lower reaction rate than that reacted with the first subset of ions to prevent over-quenching the ion mass of interest (e.g., over-quenching to z=0).
In another aspect, the invention provides an apparatus including a waveform generator which is adapted to apply a tailored waveform having at least two gaps in frequency space to an ion trap. The waveform generator typically reacts in response to a control signal from a signal generator. The apparatus also includes a source of quencher ions in fluid communication with the ion trap.
The waveform generator can be any suitable device for applying a time varying electrical potential to an ion trap. The signal generator includes any suitable device that can generate control signals for the waveform generator. For example, a computer with appropriate hardware and software could serve as both a signal generator and a waveform generator. The source of quencher ions can be any suitable source that can be adapted to be in fluid communication with the ion trap. For example, a suitable source of neutral quencher species could be a gas cylinder. Suitable sources of ionic quenchers include, but are not limited to, electron ionization, discharge, and radioactive emission sources.
In one embodiment, the apparatus further includes several memory elements. The memory elements may be portions of the random access memory of a computer, and/or discreet memory elements of a computer, the signal generator, and/or the waveform generator. In one particular embodiment, the apparatus further includes: (1) a first memory element that stores an ion mass parameter; (2) a second memory element that contains a tailored waveform generator which determines a tailored waveform having at least two gaps in frequency space based on an ion mass parameter; (3) a third memory element that stores an accumulation time parameter; (4) a fourth memory element that contains a control signal generator which determines a control signal and the length of time the control signal is applied to the waveform generator based on an accumulation time parameter; and (5) a fifth memory element that contains a parameter generator which, in response to an update signal, changes the ion mass parameter and/or the accumulation time parameter.
In another embodiment, the apparatus further includes a source of ionized molecules in fluid communication with the ion trap. Any suitable ion source can be used including, but not limited to, electrospray, laser desorption, and MALDI ion sources. In another embodiment, the apparatus further comprises a mass spectrometer in fluid communication with the ion trap. Suitable mass spectrometers include, but are not limited to, time-of-flight, quadrupole, RF multipole, Wein filter, magnetic sector, and electrostatic sector instruments.
In another aspect, the invention provides an article of manufacture where the functionality of a method of the invention is embedded on a computer-readable program means, such as, but not limited to, a floppy disk, a hard disk, an optical disk, a magnetic tape, a PROM, an EPROM, CD-ROM, or DVD-ROM.
The foregoing and other features and advantages of the invention, as well as the invention itself, will be more fully understood from the description, drawings, and claims which follow.